Summary Many cancer and stem cells exhibit a metabolic program that is distinct from most terminally differentiated cells, wherein a smaller fraction of the glycolytic product pyruvate is transported into the mitochondria and oxidized. Through a recent collaborative study, the Rutter and Thummel laboratories recently discovered the identity of the transporter, known as the Mitochondrial Pyruvate Carrier (MPC), which mediates mitochondrial pyruvate entry. Having made this breakthrough, we are now in position to test the hypothesis, first proposed about 40 years ago, that loss of the MPC underlies a portion of the decreased mitochondrial pyruvate oxidation observed in cancers. We have generated significant preliminary data in support of this hypothesis for colon cancers, and our data suggests that intestinal stem cells might exhibit the same phenomenon. We propose to employ a multi-system approach, similar to the one that enabled the initial discovery of the MPC, that uses the powerful strengths of intestinal organoid culture, Drosophila melanogaster, and mouse models of cancer and intestinal homeostasis. Utilizing this approach, we intend to test the hypothesis that mitochondrial pyruvate oxidation is necessary and sufficient to maintain appropriate stem cell homeostasis and can play an instructive role in colon oncogenesis. We will do this through three specific aims that define the mechanistic basis for the regulation and function of the MPC in instructing intestinal stem cell fate and the propensity of oncogenesis as well as define the impact of those mechanisms in intact animal models. This information will establish a new framework for understanding how core metabolic pathways can directly impact the initiation and progression of colon tumors.